Project Summary/Abstract: DESCRIPTION: Store-operated calcium entry (SOCE) is a ubiquitous signaling mechanism in eukaryotic cells crucial for mediating longer?term Ca2+ signals and restoring endoplasmic/sarcoplasmic reticulum Ca2+ after ligand induced depletion. The key operators in SOCE are the Ca2+ selective PM hexameric Orai channels (predominantly Orai1) and the ER/SR resident, dimeric transmembrane calcium sensor proteins, STIM1 and STIM2. STIM1 is activated when ER/SR luminal Ca2+ is depleted, inducing it to unfold and bind to Orai1 channels in the PM. Active Orai1 channels create discrete microdomains of high Ca2+ within ER-PM junctions that contain roughly 100-fold greater Ca2+ concentrations than resting cytoplasmic levels. New EM studies reveal Orai1 channels within ER-PM junctions are closely spaced (9-13 nm), roughly the distance spanned by the STIM-Orai Activating Regions (SOAR) of dimeric STIM1. Although clustering of Orai channels is critical for generating such Ca2+-saturated microdomains, the actual mechanism by which clustering occurs is not understood. My studies address how clustering of Orai1 channels locally controls SOCE-mediated Ca2+ signals in junctions by ?concentrating? or spatially confining Ca2+ entry through cross-linking Orai1 channels mediated by the dimeric SOAR domains in STIM1. The studies use soluble SOAR-dimers containing the critical F394H mutation that prevents SOAR binding to the Orai1 channels. I will investigate how a newly discovered and widely expressed splice variant of the STIM2 isoform (STIM2.1), containing an 8 amino acid insert adjacent to the critical F394 STIM-Orai1 binding domain in SOAR, functionally mimics the SOAR1(F394H) mutation. My aims address a critical gap in our understanding of how clustering of Orai1 spatially controls Ca2+ signals and their downstream effectors. My overall hypothesis is that STIM2.1 functions as an important negative regulator of STIM1 that prevents clustering of Orai1 channels, and controls the spatial characteristics of Ca2+ entry signals. Using new SOAR2.1 concatemeric dimers as probes, my aims are: (1) to use super-resolution microscopy to create 3D-image reconstructions of YFP-SOAR2.1 concatemer interactions with Orai1-CFP, and fluorescence recovery after photobleaching (FRAP) to assess membrane diffusion rates and size of YFP-OAR2.1 concatemers bound to Orai1; (2) to use genetically encoded Ca2+ indicators to measure localized Ca2+ signals in clusters, and a FRET-based cyclic AMP sensor, Epac2, to functionally report downstream Ca2+-dependent adenylyl cyclase (AC8) closely associated with Orai1 channels. Overall, these studies will determine how SOAR2.1 may mediate important control Orai1 channel clustering by preventing Orai1 cross-linking, and will assess how STIM2.1 may regulate Orai1 spacing and local Ca2+ microdomains mediated through downstream AC8 activity. Such studies bring together new understanding of the interdependence of local Ca2+ and cyclic AMP signals of major importance in pancreatic, neuronal, and immune cell function.